Tumor Microenvironment Stimuli‐Responsive Single‐NIR‐Laser Activated Synergistic Phototherapy for Hypoxic Cancer by Perylene Functionalized Dual‐Targeted Upconversion Nanoparticles

Abstract Although synergistic therapy has shown great promise for effective treatment of cancer, the unsatisfactory therapeutic efficacy of photothermal therapy/photodynamic therapy is resulted from the absorption wavelength mismatch, tumor hypoxia, photosensitizer leakage, and inability in intelligent on‐demand activation. Herein, based on the characteristics of tumor microenvironment (TME), such as the slight acidity, hypoxia, and overexpression of H2O2, a TME stimuli‐responsive and dual‐targeted composite nanoplatform (UCTTD‐PC4) is strategically explored by coating a tannic acid (TA)/Fe3+ nanofilm with good biocompatibility onto the upconversion nanoparticles in an ultrafast, green and simple way. The pH‐responsive feature of UCTTD‐PC4 remains stable during the blood circulation, while rapidly releases Fe3+ in the slightly acidic tumor cells, which results in catalyzing H2O2 to produce O2 and overcoming the tumor hypoxia. Notably, the emission spectrum of the UCTTD perfectly matches the absorption spectrum of the photosensitizer (perylene probe (PC4)) to achieve the enhanced therapeutic effect triggered by a single laser. This study provides a new strategy for the rational design and development of the safe and efficient single near‐infrared laser‐triggered synergistic treatment platform for hypoxic cancer under the guidance of multimodal imaging.

Chinese Academy of Sciences (China). All chemicals were used as received without any further purification.

Synthesis of NaYF 4 : Yb, Tm Upconversion Nanoparticles (UCNPs)
NaYF 4 : Yb, Tm upconversion nanoparticles were synthesized via a previously procedure with some modifications. [1] In a typical process, 1 mmol rare-earth chlorides (0.795 mmol Y 3+ , 0.2 mmol Yb 3+ and 0.005 mmol Tm 3+ ) were added to a 100 mL three-necked flask containing 6 mL oleic acid (OA) and 15 mL 1-octadecene (ODE). The solution under an argon atmosphere with vigorous stirring for 10 min, and then was heated to 170°C for 60 min before cooled down to room temperature.
Subsequently, 10 mL methanol solution containing NaOH (0.1 g) and NH 4 F (0.148 g) was added slowly and stirred for another 30 minutes. Thereafter, the solution was degassed at 110 o C for 30 min to remove the methanol and water. Then the solution was heated to 150°C and maintained for another 10 min. Then, the solution was heated to 300°C and keep for 90 min under an argon atmosphere. The nanoparticles were precipitated with ethanol, washed with cyclohexane/ethanol (1:2, v/v) three times, and then re-dispersed in cyclohexane.
In order to enable the nanoparticles to absorb 808 nm laser, we coated a NaYF 4 : 20% Nd shell onto the above nanoparticles. Generally, 0.06 mmol NdCl 3 , 0.24 mmol YCl 3 , were added into a 100 mL three neck round bottom flask containing 5 mL OA and 6 mL ODE. The solution was heated to 110°C and maintained for 10 min under an argon atmosphere with stirring. Then it was heated to 170°C and kept for 60 min before cooling down. When the temperature of the solution was lowered to 60°C, 20 mg of NaYF 4 : 0.05% Tm 3+ , 20% Yb 3+ nanoparticles was added, and heated to 150°C, the subsequent process is the same as the above operation to obtain the final upconversion nanoparticles.

Ligand-Exchange Reactions of UCNPs
Briefly, 5 mL of dichloromethane solution of NOBF 4 (0.01 M) was added to 5 mL of UCNPs dispersion in hexane (5 mg/mL) at room temperature. The resulting mixture was shaken gently and sonicated in an ice-water bath until the precipitation of UCNPs was observed. After being washed 3 times with absolute ethanol, the precipitated UCNPs were re-dispersed in water.

Preparation of UCNP@TA/Fe
The nanofilm was conformally coated according to previous reports. [2] The 5 mg UCNPs were added to 1 mL of natural polyphenol tannic acid (TA) solution (3.2 mg/mL), followed by 10 min of ultrasound. Then, 0.5 mL FeCl 3 solution (1.6 mg/mL) was added under vigorous magnetic stirring or fast stirring for 1 min at ambient temperature, and the mixture turned black immediately. Afterwards, they were centrifuged quickly and rinsed twice with 75% ethanol and once with deionized water to obtain UCNP@TA/Fe.

Preparation of UCNP@TA/Fe-TPP-cRGD (UCTTD)
In order to give the UCNP@TA/Fe targeting ability, mitochondrial targeting molecule TPP and RGD peptides that recognize the overexpressed αvβ 3   After stirring in the dark for 2 h, the activated TPP was added to 1 mL of PEG-modified UCNP@TA/Fe mentioned above. After overnight reaction at room temperature, the UCNP@TA/Fe-TPP was collected by centrifugation and washed with deionized water for 3 times. In order to modify the cRGD targeting protein, UCNP@TA/Fe-TPP was added to the cRGD solution (0.05 mg/mL), rotated at 4°C for 12 h, and then washed with deionized water for 3 times to obtain the UCNP@TA/Fe-TPP-cRGD (UCTTD).

Loading and Release Experiment of the Photosensitizer
To investigate the photosensitizer (PC4) loading and release behavior, 1 mg UCTTD was added to 1 mL of PC4 solution with different concentrations (25, 50, 100, 150, or 200 µg/mL) and constantly shaken in the dark for 36 h at room temperature. The concentration of PC4 (C x ) in the supernatant was measured via UV-vis absorption at 6, 12, 24 and 36 h and calculated by the equation: Loading rate = (C 0 -C x ) /C 0 × 100%, where C 0 is the initial concentration of PC4. For the release behavior of PC4, UCTTD-PC4 was placed in PBS solution with pH of 5.0, 6.5 and 7.4, respectively, and the concentration of PC4 (C x ) released in the supernatant was measured at 1, 3, 6, 12, 24 and 48 h (Release rate = C x /C 0 × 100%, where C 0 is the initial amount of PC4 has been loaded on UCTTD).

Characterization of Nanomaterials
The HR-TEM images were recorded by a high resolution transmission electron microscope operating at 200 kV (JEM-2100F, Japan). HAADF-STEM images, elemental mappings and energy dispersion X-ray spectra were obtained by high resolution electron microscopy. The surface morphologies of nanocarriers were characterized by scanning electronic microscopy (SEM, XL30 ESEM, Japan). Fourier Transform Infrared (FT-IR) spectra (VERTEX70 FT-IR Spectrophotometer, Bruker Optics, Germany) was employed to confirm the connection of the targeted molecules.
The absorption spectra were measured with a Cary500 Scan UV-vis scanning spectrophotometer (Varian, USA). The fluorescence spectra were carried out on a FLWOROMAX-4 fluorescence spectrometer (PL) (HORIBA). Hydrodynamic diameter and zeta potential were acquired by a Malvern Zetasizer Nano ZS (UK).
Meanwhile, the UCTTD solution was irradiated by different laser power (0.1, 0.3, 0.5, 0.7, and 0.9 W/cm 2 ). Moreover, to investigate the photothermal stability, the UCTTD solution was irradiated six cycles of consecutive laser on/off under similar conditions. The thermocouple probe linked to a digital thermometer (TES K-Type Thermometer 1319A, Taiwan) and an infrared thermal camera (FLIR C2, USA) recorded the real time temperature and infrared thermal images every 30 s. The photothermal conversion efficiency (η) was calculated by the previous method. [3]

Oxygen Production in Vivo and in Vitro
The extracellular generation of O 2 was measured by YSI5000 dissolved oxygen-BOD

Intracellular ROS Detection
The intracellular generation of ROS was studied with DCFH-DA Kit by CLSM imaging. The Capan-1 cells were seeded into 96-well plates at the density of 1 × 10 4 per well. After 12 h of incubation, cells were treated with PBS, UCTTD, PC4 or UCTTD-PC4 for 12 h before washed with PBS, then, the ROS sensor (0.1 µM DCFH-DA) was added to each well. The cells were then incubated for another 20 min before irradiated with an 808 nm laser (0.3 W/cm 2 ) or 470 nm laser (0.08 W/cm 2 ) for 10 min. Finally, the intracellular distribution of DCF fluorescence, which represents ROS was recorded via CLSM after washing with PBS.

Targeted Delivery
The cultured cells were divided into two groups and co-incubated with UCNP@TA/Fe-TPP-PC4 (UCTT-PC4) and UCTTD-PC4 with cRGD-targeted protein for 6 h respectively. After digestion, the cells were quantitatively analyzed by flow cytometry (FCM).

Cytotoxicity Assay
Cell activity and toxicity tests were evaluated via CCK-8 kit. The Capan-1 cells were incubated overnight, and then different concentrations of UCTTD (0, 150, 300, 600, 900, 1200, 1500, 2000 µg/mL) were added to text the toxicity of the UCTTD. Another, different formulations (PBS, UCTTD, PC4, UCTTD-PC4) were added and incubated for 6 h, followed by the irradiation of the 808 nm laser (0.3 W/cm 2 ) or 470 nm laser (0.08 W/cm 2 ) for 10 min. After continuing to incubate for 24 h and 48 h, the cells were washed with PBS, and then added 100 μL of fresh culture medium (containing 10 μL of CCK-8 reagent). Finally, the microplate reader (Spark Control TM Tecan, USA) recorded the absorbance at 450 nm. The experiment was repeated three times for each group.

Cell Apoptosis Assay
The Capan-1 cells were seeded in 48-well plates and incubated overnight, and then the cells were treated with PBS, UCTTD, PC4, or UCTTD-PC4 respectively for 6 h, followed by the irradiation of the 808 nm laser (0.3 W/cm 2 ) or 470 nm laser (0.08 W/cm 2 ) for 10 min. After continuing to incubate for another 42 h, the cells were collected and washed with PBS. Finally, the apoptosis detection kit was used to evaluate cell apoptosis. The fluorescence of 20, 000 cells were detected using a flow cytometer (BD AccuriTM C6, USA). Non-treated cells were used as the control.

Animal Tumor Model
The tumor models were established on female BALB/c nude mice of 5-6 weeks old

Biodistribution Assay
The tumor-bearing mice were randomly divided into 4 groups (n = 3 per group) after the tumor volume reached approximately 200-300 mm 3 to study the biodistribution behavior, the mice were intravenously injected with PBS, PC4, UCTTD or UCTTD-PC4 (5 mg/mL), respectively. Afterward, the main organs such as heart, liver, spleen, lung, kidney, as well as tumors were harvested at 6 and 24 h post-injection respectively. The fluorescence intensity was imaged by Davinch Invivo HR imaging system (Davinch K, Korea). Subsequently, the tumors and other healthy organs were lyophilized, weighed and dissolved for ICP-MS (X Series 2, Thermo Scientific, USA) analysis of Y content.

Photoacoustic Imaging and Photothermal Imaging
The photoacoustic imaging (PAI) capability of the UCTTD was verified by the multispectral optoacoustic tomography imaging system (MSOT inVision 128, Finally, the results of PA images were recorded by MOST system. For in vivo photothermal imaging, when the tumor-bearing mice were injected intravenously with 200 μL UCTTD (5 mg/mL) at different time points, the mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg), followed by irradiation of 808 nm laser (1.2 W/cm 2 ). The infrared thermal imaging camera was employed to monitor the tumor temperature and collect photothermal images.

In Vitro and in Vivo Magnetic Resonance Imaging (MRI)
In order to measure the relaxation rate of UCTTD, the sample was diluted to 0, 0.5, 1, 2, or 4 mg/mL for in vitro magnetic resonance imaging (MRI). For in vivo MRI imaging, when the tumor volume of nude mice reached 200-300 mm 3 , 200 µL UCTTD (5 mg/mL) was injected intravenously into the mice. At 0 h and 24 h, the mice were anesthetized, followed by 3.0 T CX extremely fast nuclear magnetic resonance (Philips Ingenia) imaging system collects T1-and T2-weighted imaging pictures.

Upconversion Luminescence Imaging (UCL)
Upconversion luminescence imaging was achieved by Davinch Invivo HR imaging system equipped with 808 nm laser. Briefly, when the tumor volume of nude mice reached 100 mm 3 , the mice were anesthetized and 50 µL UCTTD was injected intratumoral. Upconversion luminescence signals were recorded in the green channel.

Photothermal and Photodynamic Synergistic Therapy
When the tumor volume of female BALB/c nude mice reached 100 mm 3  The mice were euthanized at 2 weeks after treatments and then the blood was collected for biochemistry analysis. The tumors and organs (liver, heart, spleen, lung, and kidney) in each group were isolated from the mice, and then fixed with 4% neutral buffered formalin and embedded in paraffin for hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Ki-67 and HIF-1α labeling. Finally, they were examined by the inverted fluorescence microscope (NIKON, Japan).

Statistical Analysis
All data were presented as mean ± standard deviation (SD) of triplicates for cell experiments or quintuplicates for animal experiments unless otherwise indicated. The differences in experimental data were compared by the One-way ANOVA statistical analysis by the SPSS software (IBM SPSS Statistics, USA). The level of statistical significance was settled at *p < 0.05. **p < 0.01; ***p < 0.001.       Data are presented as means ± SD. L 470 represents 470 nm laser at power density of 0.08 W/cm 2 for 10 min, L represents 808 nm laser at power density of 1.2 W/cm 2 for 10 min.